Fatty acids bearing chemical modifications in addition to the common double bonds are found in the storage lipids of many oilseeds (Badami and Patil (1981) Prog. Lipid Res. 19:119-153). Some of these modifications functionalize the fatty acid to produce products that are useful in industrial applications; this is an alternative to the more common usage of plant-derived lipids as foods. Examples are the use of the hydroxylated fatty acid ricinoleic acid in lubricants, and the short- or medium-carbon chain length fatty acids from palm oil in detergents. In some cases, fatty acid composition of the storage lipids of oilseeds produced in temperate climates can be modified by the addition of genes from exotic sources so that large amounts of unique fatty acids are produced (Ohlrogge, J. B. (1994) Plant Physiol. 104, 821-826).
Fatty acids containing conjugated double bonds are major components of the seed oil of a limited number of plant species. For example, calendic acid (8-trans, 10-trans, cis-12-octadecatrienoic acid) composes greater than 50% of the total fatty acids of the seed oil of Calendula officinalis (Crombie and Holloway (1984) J. Chem. Soc. Chem. Commun. 15, 953-955, Chisholm, M. J. & Hopkins, C. Y. (1967) Can. J. Biochem 45:251-254). Another example, α-parinaric acid (9-cis, 11-trans, 13-trans, 15-cis-octadecatetraenoic acid) and β-parinaric acid (9-trans, 11-trans, 13-trans, 15-cis-octadecatetraenoic acid) compose more than 25% of the total fatty acids of the seed oil of Impatiens species (Bagby, M. O., Smith, C. R. and Wolff, I. A. (1966) Lipids 1, 263-267). In addition, α-eleostearic acid (9-cis, 11-trans, 13-trans-octadecatrienoic acid) and β-eleostearic acid (9-trans, 11-trans, 13-trans-octadecatrienoic acid) compose >55% of the total fatty acids of the seed oil of Momordica charantia (Chisolm, M. J. and Hopkins, C. Y. (1964) Can. J. Biochem. 42, 560-564; Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). Calendic acid and eleostearic acid are both 18:3 fatty acids, like linolenic acid, however, their structures are quite different, as shown in FIG. 1. Another fatty acid containing conjugated double bonds is found in the seeds of Dimorphotheca sinuata. This unusual C18 fatty acid, dimorphecolic acid (9-OH-18:2Δ10trans,12trans), contains two conjugated trans-double bonds between the Δ10 and Δ11 carbon atoms and between the Δ12 and Δ13 carbon atoms as well as a hydroxyl group on the Δ9 carbon atom [Binder, R. G. et al, (1964) J. Am. Oil Chem. Soc. 41:108-111; Morris, L. J. et al., (1960) J. Am. Oil Chem. Soc. 37:323-327]. Thus, there are certain 18:2 and 18:3 plant fatty acids that contain conjugated double bonds.
The presence of conjugated double bonds in fatty acids provides the functional basis for drying oils such as tung oil that are enriched in isomers of eleostearic acid. This is due largely to the fact that fatty acids with conjugated double bonds display high rates of oxidation, particularly when compared to polyunsaturated fatty acids with methylene interrupted double bonds. Drying oils, such as tung oil, are used as components of paints, varnishes, and inks.
Conjugated fatty acids can also be used as an animal feed additive. Conjugated linoleic acids (CLAs, 18:2) have been used to improve fat composition in feed animals.
U.S. Pat. No. 5,581,572, issued to Cook et al. on Dec. 22, 1998, describes a method of increasing fat firmness and improving meat quality in animals using conjugated linoleic acids.
U.S. Pat. No. 5,554,646, issued to Cook et al. on Sep. 10, 1996, describes a method of reducing body fat in animals using conjugated linoleic acids.
U.S. Pat. No. 5,519,451, issued to Cook et al. on Jul. 6, 1999, describes a method of improving the growth or the efficiency of feed conversion of an animal which involves animal feed particles having an inner core of nutrients and an outer layer containing a conjugated fatty acid or an antibody that can protect the animal from contacting diseases that can adversely affect the animal's ability to grow or efficiently convert its feed into body tissue.
U.S. Pat. No. 5,428,072, issued to Cook et al. on Jun. 27, 1995, describes a method of enhancing weight gain and feed efficiency in animals, which involves the use of conjugated linoleic acid.
The mechanism by which these effects are realized is not known. It is believed that no one heretofore has discussed the use of conjugated 18:3 fatty acids (conjugated linolenic acids or ClnAs), for improving animal carcass characteristics.
The biosynthesis of fatty acids with conjugated double bonds is not well understood. Several reports have indicated that conjugated double bonds are formed by modification of an existing double bond (Crombie, L. and Holloway, S. J. (1985) J. Chem. Soc. Perkins Trans. I 1985, 2425-2434; Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). For example, the double bonds at the 11 and 13 carbon atoms in eleostearic acid have been shown to arise from the modification of the Δ12 double bond of linoleic acid (18:2Δ9,12) (Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). The exact mechanism involved in conjugated double formation in fatty acids, however, has not yet been determined. Fatty acid desaturase (Fad)-related enzymes are responsible for producing 18:3 Δ9,11,13 oils such as α and β-eleostearic acid and 18:4Δ9,11,13,15 oils such as α and β-parinaric acid in Impatiens, Momordica, and Chrysobalanus. Insertion of a chimeric gene comprising an isolated nucleic acid fragment encoding these enzymes into species that do not normally accumulate conjugated double-bond containing fatty acids resulted in production of eleostearic and/or parinaric acids (Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96:12935-12940; and WO 00/11176, published on Mar. 2, 2000, the disclosure of which is hereby incorporated by reference). The present invention extends this work by answering whether 18:3 Δ8,10,12 fatty acids like calendic or dimorphecolic acids can also be produced in transgenic plants. Unlike the Fad-related enzymes that modify the delta-12 position to produce eleostearic and parinaric acids, the enzymes of the present invention (with one exception as is discussed below with respect to DMFad2-1) modify the delta-9 position of fatty acids to produce calendic and dimorphecolic acids. One enzyme is disclosed herein which is associated with the formation of a trans-delta-12 double bond. The product of this enzymatic reaction then becomes the substrate for a reaction involving conjugated double bond formation comprising a delta-9 position of fatty acids. Isolation and characterization of two Calendula cDNAs, two Dimorphotheca cDNAs, and expression of a chimeric transgene, are described herein.